It is assumed that hepatocellular bile formation depends on at least two mechanisms. One fraction (BSDF) depends on transcellular movement of bile salts while the other, termed bile salt independent fraction (BSIF) is thought to depend on sodium excretion. Sodium, chloride and water could move from sinusoid to canaliculus across the cell, between cells or both. It is unknown by which pathway water and sodium reach the canaliculus. The objective of the proposed research is therefore, to quantitate water and ion transport via the transcellular and paracellular pathway. Water and ion fluxes will be studied in the in situ perfused rat liver by use of a multiple indicator dilution technique. Biliary dilution curves of labeled sodium, chloride and water will be compared to the dilution curve of 14-taurocholate as an intracellular reference substance. Bile salt dependent and independent fractions of bile flow will be stimulated by bile salt infusion and phenobarbital induction, respectively. The regulatory influence of the tight junction on the paracellular pathway will be investigated by altering calcium concentration in the perfusate and by inhibiting microfilaments with cytochalasin B. Behaviour of ion and water transport across the trans- and paracellular pathway will be studied also in different models of intra- and extrahepatic cholestasis. Ethinylestradiol and taurolithocholate will be used to induce intrahepatic cholestasis, while alphanaphthylisothiocyanate and bile duct ligation will be used to induce extrahepatic cholestasis. These studies will permit for the first time quantitation of water flow across the trans- and paracellular pathway under physiological and pathological conditions. Furthermore they will allow insight into the mechanism whereby BSIF if formed by studying regulation of sodium and chloride transport.